Pathogenic west african human immunodeficiency virus type 2 (HIV-2) group F isolate

ABSTRACT

The claimed invention is directed toward an HIV-2 isolate designated NWK08F, including variants, and isolated proteins and nucleotides obtained from said isolates.

This is a U.S. National Phase Application of PCT/US2009/064349, filedNov. 13, 2009, which claims the benefit of priority to U.S. ProvisionalPatent Application No. 61/114,807, filed Nov. 14, 2008, and to U.S.Provisional Patent Application No. 61/163,190, filed Mar. 25, 2009, allof which are incorporated herein by reference in their entireties.

DESCRIPTION OF THE INVENTION

Substantial progress has been made in our understanding of acquiredimmune deficiency syndrome (AIDS). The principal causative agent of AIDSis human immunodeficiency virus (HIV), a non-transforming retroviruswith a tropism for CD4 T-helper cells. AIDS is characterized by aprogressive depletion of the CD4 T cell population with a concomitantincreasing susceptibility to the opportunistic infections that arecharacteristic of the disease. Epidemiological studies indicate thatHIV-1 is the etiological agent responsible for the majority of AIDScases and these infections are widely spread throughout the world.

A second type of HIV, HIV-2, has been isolated from patients in WestAfrica, but has not appreciably spread beyond this area. The incidenceof HIV-2 infection has declined over the last 16-20 years (Hamel et al.,AIDS Res Hum Retroviruses 23:1189-96 (2007); Van der Loeff et al., Int JEpidemiol 35:1322-28 (2006)). There are at least 8 known subtypes ofHIV-2, referred to as subtypes A-H. The majority of human HIV-2infections are caused by subtypes A and B, which are known as theepidemic subtypes. Only a small percentage of individuals infected withHIV-2 subtypes A and B develop the immunodeficiency characteristic ofAIDS (Marlink et al., Science 265:1587-90 (1994). Infections with thenon-epidemic HIV-2 subtypes C-G are generally known only as singleperson infections, and have not been shown to lead to immunodeficiency(Gao et al., J Virol 68:7433-47 (1994); Chen et al., J Virol 71:2953-60(1997)). There is also only one known case of a person infected withHIV-2 subtype H, and this virus caused immunodeficiency in the infectedman from the Ivory Coast (Damond et al., AIDS Res Hum Retroviruses20:666-72 (2004)).

HIV-2 subtype F was discovered 16 years ago in an individual from SierraLeone (Chen et al., J Virol 71:2953-60 (1997)). Despite repeatedattempts, live virus was never isolated from the infected patient andthe patient remained healthy during the time of observation.

We have now identified a new strain of HIV-2. It is accordingly aprimary object of the invention to provide a novel strain of Subtype FHIV-2, nucleic acids and polypeptides derived from this virus, andmethods of detecting the presence of this novel virus in patients andcell culture.

In one embodiment, the invention provides an isolated HIV-2 viruscomprising the virus deposited at the American Type Culture Collection(“ATCC”) (ATCC, 10801 University Boulevard. Manassas, Va. 20110-2209) asHIV-2NWK08F. In some embodiments, this HIV-2 virus comprises thenucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3. In an additionalembodiment, the HIV-2 virus is a variant of HIV-2NWK08F, wherein thevariant comprises a nucleotide sequence with 95% homology to SEQ ID NO:1or SEQ ID NO:3.

In one embodiment, the invention provides a nucleic acid comprising anucleotide sequence of SEQ ID NO:1 or SEQ ID NO:4, or fragments thereof.This nucleic acid may comprise at least 6 contiguous nucleotides of SEQID NO:1 or SEQ ID NO:4. In some embodiments, the nucleic acid contains adetectable label.

In other embodiments, the invention provides a polypeptide comprising anamino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, or SEQ IDNO:6, or fragments thereof. This polypeptide may comprise at least 6contiguous amino acids of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, or SEQID NO:6. In particular embodiments the polypeptide encodes the Env, Pol,Gag, or Nef protein of HIV-2NWK08F.

In another embodiment, the invention provides an antibody thatspecifically binds to a polypeptide derived from the HIV-2NWK08F virus.In some embodiments, the antibody specifically binds to an Env, Pol,Gag, or Nef polypeptide of HIV-2NWK08F, but does not bind to Envpolypeptides from other HIV-1 or HIV-2 virus strains.

In another embodiment, the antibody specifically binds to an Env, Pol,Gag, or Nef polypeptide of HIV-2 subtype F, but does not bind to Envpolypeptides from other HIV-1 or HIV-2 virus strains or subtypes.

The invention also provides compositions and methods for detectingHIV-2NWK08F nucleic acids and polypeptides. In one embodiment, theinvention provides a composition for detecting HIV-2NWK08F nucleic acidscomprising at least 6 contiguous nucleotides of SEQ ID NO:1 or SEQ IDNO:4. In another embodiments, the invention provides a method fordetecting HIV-2NWK08F nucleic acids in a biological sample comprising

(a) contacting the biological sample with a composition comprising anucleotide sequence of HIV-2NWK08F; and

(b) detecting the hybridization of the HIV-2NWK08F nucleotide sequenceto the nucleic acid of the biological sample.

In some embodiments, the methods for detecting HIV-2NWK08F nucleic acidsinvolve the amplification of HIV-2NWK08F nucleic acids prior to orduring the detection step.

In one embodiment, the invention provides a method of detectingHIV-2NWK08F nucleic acids in a biological sample comprising

(a) contacting the biological sample with primers derived fromHIV-2NWK08F capable of amplifying an HIV-2NWK08F genome;

(b) amplifying the HIV-2NWK08F nucleic acid; and

(c) detecting the presence of amplified HIV-2NWK08F nucleic acid.

In another embodiment, the invention provides a composition fordetecting HIV-2NWK08F polypeptides comprising an antibody specific forthose polypeptides. In an exemplary embodiment, the invention provides amethod for detecting HIV-2NWK08F polypeptides in a biological samplecomprising

(a) contacting the biological sample with a composition comprising atleast one anti-HIV-2NWK08F antibody; and

(b) detecting an immunological complex formed between the polypeptideand the antibody used.

The invention also provides compositions and methods of detectingantibodies specific for HIV-2NWK08F. In one embodiment, the inventionprovides compositions for detecting antibodies specific for HIV-2NWK08Fcomprising at least 6 contiguous amino acids of SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:5, or SEQ ID NO:6. In another embodiment, the inventionprovides a method for detecting antibodies against HIV-2NWK08F virus ina biological sample comprising:

(a) contacting the biological sample with a composition comprising atleast one antigen of the HIV-2NWK08F virus; and

(b) detecting an immunological complex formed between theanti-HIV-2NWK08F antibodies and the antigen used.

In another embodiment, the invention provides a method for thepreparation of hybridomas which produce monoclonal antibodies specificfor the HIV-2NWK08F Env of Gag polypeptide, comprising the following thesteps:

(a) immunizing a mammal with a polypeptide from the HIV-2NWK08F Env orGag polypeptide or immunogenic fragment thereof;

(b) isolating immunized splenocytes from said mammal;

(c) fusing the immunized splenocytes with a myeloma cell line to producehybridomas;

(d) selecting for the hybridomas by culturing in selective media;

(e) clonally expanding the hybridomas in appropriate culture media; and,

(f) identifying and characterizing those hybridomas that producemonoclonal antibodies specific for HIV-2NWK08F Env, Pol, Gag, or Nefpolypeptide.

In yet another embodiment, the invention provides methods of treatingpatients infected with HIV-2NWK08F by administering anti-viral drugs.

Additional objects and advantages of the invention will be set forth inpart in the description which follows, and in part will be obvious fromthe description, or may be learned by practice of the invention. Theobjects and advantages of the invention will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a phylogenetic tree showing the relationship betweenHIV-2NWK08F env gene sequences to other HIV and SIV strains.

FIG. 2 is a phylogenetic tree showing the relationship betweenHIV-2NWK08F gag gene sequences to other HIV and SIV strains.

BRIEF DESCRIPTION OF THE SEQUENCES

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Nov. 12, 2009, is named09932000.txt, and is 25,235 bytes in size.

SEQ ID NO:1 is a partial nucleotide sequence of the env and nef genes ofHIV-2NWK08F.

SEQ ID NO:2 is a partial amino acid sequence of the Env polypeptide ofHIV-2NWK08F.

SEQ ID NO:3 is a partial amino acid sequence of the Nef polypeptide ofHIV-2NWK08F.

SEQ ID NO:4 is the full nucleotide sequence of gag gene of HIV-2NWK08Fand a partial nucleotide sequence of the 5′-LTR and the pol gene ofHIV-2NWK08F.

SEQ ID NO:5 is the full amino acid sequence of the Gag polypeptide ofHIV-2NWK08F.

SEQ ID NO:6 is a partial amino acid sequence of the Nef polypeptide ofHIV-2NWK08F.

SEQ ID NOs:7-20 are primers derived from the env/nef genes of HIV-2subtype F virus.

SEQ ID NOs:21-36 are primers derived from the gag/pol gene of HIV-2subtype F virus.

SEQ ID NOs:37-40 are primers derived from the env gene of HIV-2 subtypeF virus.

SEQ ID NOs:41-44 are primers derived from the gag gene of HIV-2 subtypeF virus.

SEQ ID NOs:45 and 46 are primers derived from the env gene ofHIV-2NWK08F for use in a real time PCR assay for HIV-2NWK08F viral load.

SEQ ID NO:47 is a probe derived from the env gene of HIV-2NWK08F for usein detection of the PCR products in a real time PCR assay forHIV-2NWK08F viral load.

DESCRIPTION OF THE EMBODIMENTS

The invention is based, in part, on the discovery of a novel strain ofHIV-2 in a patient in Newark, N.J. The invention is further based, inpart, on the isolation of an HIV-2 virus, referred to as HIV-2NWK08F,from the patient. The invention is also based, in part, on the nucleicacid sequence of this virus, and in particular, the nucleic acidsequence of the genes encoding the Gag and Env polypeptides of thisvirus.

Patient X, a 68 year old man, moved from his homeland of Sierra Leone toNew Jersey in 2007. During the immigration process, he tested positivein a general screen for antibodies to HIV. Upon further investigation,Patient X repeatedly tested positive for antibodies in an assay thatdetected antibodies against either HIV-1 or HIV-2. However, thepatient's blood tested negative for HIV-1 by Western blot and PCRassays. His HIV-1 viral load was below the lower limit of the assay (aquantitative RT-PCR assay from LabCorp). Interestingly, the patienttested positive for HIV-2 antibodies, but a PCR assay for HIV-2 proviralDNA was negative, indicating that the patient may have been infectedwith an unidentified strain of HIV-2.

Patient X had a CD4 T-cell count of 338 cells/μl and a CD4:CD8 ratio of0.52. These results are indicative of CD4 T-cell lymphopenia. Thecombination of the results from the HIV testing and the patient'sreduced CD4 T-cell count suggested that Patient X was actively infectedwith an unrecognized strain of HIV-2.

Co-culture of Patient X's peripheral blood mononuclear cells (PBMCs)with PHA-stimulated normal donor PMBCs or CEM-X-174 cells resulted inviral isolation. Portions of the env and gag genes of the provirusproduced by these cells were successfully amplified by PCR with primersderived from an HIV-2 subtype F strain (Chen et al., J Virol71:3953-3960 (1997), incorporated by reference herein). The amplifiedregions were sequenced and a real-time PCR protocol was developed withprimers derived from the env gene of the new virus to quantify viralload. Two recent studies of HIV-2 infected individuals found the medianproviral load to be ˜300 copies per 10⁶ PBMC (Gottleib et al., AIDS22:1379-80 (2008); Popper et al., J Virol 74:1554-57 (2000)). Incontrast, Patient X had a proviral load of 6,100 copies per 10⁶ PBMC.

The sequenced regions of the new virus were subjected to phylogeneticcomparisons to existing HIV strains (Posada, et al., Syst Biol50:580-601 (2001); Swofford, PAUP*. Phylogenetic analysis usingparsimony (*and other methods), version 4. Sinauer Associates,Sunderland, Mass. (1999)). HIV-2NWK08F clustered significantly with fourother viruses, all from Sierra Leone. Two of the viruses (strains ofHIV-2 subtypes E and F) have not been known to cause immune suppression,nor have they been shown to be transmitted from person to person. Theother 2 viruses were simian immunodeficiency viruses (SIVs) found insooty mangabey monkeys in Sierra Leone, documenting transmission frommonkey to humans in the area. It is unknown how Patient X acquired thevirus. Since he denies exposure to monkeys and has not received blood orblood product transfusions, he most likely contracted the virus fromanother person.

Accordingly, the invention provides a new strain of HIV-2 virus, thenucleotide sequence of the virus, viral proteins and fragments thereof,methods of detecting HIV-2 virus infection, and methods of treating apatient infected with HIV-2.

Viruses

The invention provides an isolated virus, known as HIV-2NWK08F.HIV-2NWK08F was deposited with the American Type Culture Collection(“ATCC”) (ATCC, 10801 University Boulevard. Manassas, Va. 20110-2209)according to the provisions of the Budapest Treaty. All restrictions onthe availability to the public of the above ATCC deposit will beirrevocably removed upon the granting of a patent on this application.In another embodiment, the invention provides variants of this viruscomprising a nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3 or asequence substantially homologous to SEQ ID NO:1 or SEQ ID NO:3, i.e.,at least about 85%, about 90% or about 95% homologous at the nucleotidelevel. In yet another embodiment, the invention provides variants of thevirus that encodes polypeptides comprising an amino acid sequence of SEQID NO:2 or SEQ ID NO:4 a sequence substantially homologous to SEQ IDNO:2 or SEQ ID NO:4, i.e., at least about 85%, about 90% or about 95%homologous at the amino acid level. The invention also provides cells ormammals (including humans) infected with an isolated HIV-2NWK08F virusor variant.

The invention also relates to HIV-2 variants characterized by thenucleotide sequences of the HIV-2NWK08F virus deposited at the ATCC, aswell as related variants that comprise a nucleotide sequence that issubstantially homologous to the sequences of the virus deposited at theATCC as HIV-2NWK08F, i.e., at least about 85%, 90% or 95% homologous atthe nucleotide level. The sequences of HIV-2NWK08F and its variantsdisclosed herein characterize a new HIV-2 virus that is part of asubclass of HIV-2 viruses, currently known to be found primarily inSierra Leone.

“Purified” or “isolated” HIV isolate refers to a preparation of HIVvirus particles which has been isolated from the cellular constituentswith which the virus is normally associated, and from other types ofviruses which may be present in the infected tissue. The techniques forisolating viruses are known to those of skill in the art, and include,for example, centrifugation and affinity chromatography.

An HIV “particle” is an entire virion, as well as particles which areintermediates in virion formation. HIV particles generally have one ormore HIV proteins associated with the HIV nucleic acid.

Nucleic Acids

The invention provides a nucleic acid comprising a nucleotide sequenceof HIV-2NWK08F. In some embodiments, the nucleic acid is RNA. In otherembodiments, the nucleic acid is DNA. In particular embodiments, thenucleic acid is isolated viral RNA or proviral DNA. In otherembodiments, the nucleic acid is a cDNA molecule. In other embodiments,the nucleic acid is an oligonucleotide. The nucleic acid may benaturally-occurring, non-naturally occurring, recombinantly produced, orsynthetic.

The nucleic acid molecules of the present invention may be used, e.g.,(1) to produce HIV-2NWK08F polypeptides; (2) as probes in nucleic acidhybridization assays; (3) as primers for reactions involving thesynthesis of HIV-2NWK08F nucleic acid; (4) as binding partners forseparating HIV-2NWK08F viral nucleic acid from other constituents whichmay be present; (5) as a component of a partial or complete HIV-2NWK08Fvirion; and (6) as anti-sense nucleic acid for preventing thetranscription or translation of viral nucleic acid.

In some embodiments, the nucleic acid comprises the entire viral genome.In other embodiments, the invention provides a nucleic acid comprisingsequences that encode the Env, Nef, Gag, or Pol protein of HIV-2NWK08F.In particular embodiments, the invention provides a nucleic acidcomprising all or a portion of SEQ ID NO:1 or SEQ ID NO:4 or theircomplements. In particular embodiments, the invention provides a nucleicacid that encodes all or a portion of the amino acid sequence of SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:6.

TABLE 1  HIV-2NWK08F Sequences Env/Nef Nucleotide Sequence: SEQ ID NO: 1TACCTTCATGTGGACAAATTGCAGAGGAGAATTTTTATATTGTAAAATGAATTGGTTCCTAAATTGGGTAGAAGACAGAAATATAACTCATGGAAGATGGAGTACTCAAAAACCAGCAGAGAAACAGAAGAGGAACTATGTGCCTTGCCACATAAGGCAAATCATAAATACTTGGCACAAAGTAGGGAAAAATGTGTACCTGCCTCCAAGAGAAGGTAATCTAACGTGTAACTCATCAGTAACAAGCATAATTGCAAACATAGACTGGACAAGCGACAATGAAACTAATATCACCATGAGTGCAGAAGTGGCAGAACTGTATCGATTAGAGTTGGGTGACTATAAATTAGTAGAGATAACACCAATTGGCTTGGCCCCAACAGAAGTAAAAAGATATTCCTCAGCAACACCGAGGAATAAGAGAGGGGTCTTTGTGCTAGGGTTCTTGGGATTTCTCGCAACGGCAGGTTCTGCAATGGGCGCAGCGTCGCTGACGCTGACAGCTCAGTCTCGGACTTTACTGGCTGGGATAGTGCAGCAACAGCAGCAGCTGTTGGATGCAGTCAAGAGACAACAAGAATTGTTGCGATTGACAGTCTGGGGGACTAAAAACCTCCAGACACGCGTCACTGCCATCGAGAAATACCTAAAGGATCAGGCACAGCTAAATTCATGGGGGTGTGCATTTAGACAGGTCTGCCATACTACTGTACCATGGCCAAATGACACATTGCAACCAAATTGGGACAACATGACTTGGCAAGAGTGGGAAAGGAAAGTAGACTTTCTCACAGAAAACATCACAGAACTCTTGGAGCAGGCACAGATTCAACAAGAAAAAAATATGTATGAACTACAAAAATTGAACAGCTGGGATGTGTTTGGCAATTGGTTTGACCTCAGCTCCTGGATCACCTACATACAGTATGGAGTATACTTAGTAGTAGGAGTAATAGGGCTTAGAATAAGTATATATATAGTACAGATGCTATTGAGGCTTAGAAAGGGCTATAGGCCCGTGTTCTCTTCCCCACCCTCTTATCGCCAGCAGATCCATATCCGACGGGACCAGGAACTGCCAGACGGAGAAGACAGAGAAGAAGACGGTGGAGAAAAAGGTGGCAACAGATCCTGGCCCTGGCAGATAGAGTACATTCATTTCCTGATCCGCCAGTTGATTCGCCTCTTGACTTGGCTATACAGCAATTGCAGAGACTTAATATACAAGAGCTTCCAGACCCTCCACCAGCTGACCAGTGCAGCAGCAACAGCAACTAGAGACTTTATCAGAACAGAAGCCAGTTACATCAGCTATGGGTGGCAATACTTCCTCGAAGCCCTCCAAGCGGCAATGCAGACTGCGGGAGAGACTCTTGCAAGCGCGGGGGGAGAATTATGGGCAACTCTGGGAAGGATT (SEQ ID NO: 1)Env Amino Acid Sequence: Encoded by nucleotides 8-1417 of SEQ ID NO: 1MWTNCRGEFLYCKMNWFLNWVEDRNITHGRWSTQKPAEKQKRNYVPCHIRQIINTWHKVGKNVYLPPREGNLTCNSSVTSIIANIDWTSDNETNITMSAEVAELYRLELGDYKLVEITPIGLAPTEVKRYSSATPRNKRGVFVLGFLGFLATAGSAMGAASLTLTAQSRTLLAGIVQQQQQLLDAVKRQQELLRLTVWGTKNLQTRVTAIEKYLKDQAQLNSWGCAFRQVCHTTVPWPNDTLQPNWDNMTWQEWERKVDFLTENITELLEQAQIQQEKNMYELQKLNSWDVFGNWFDLSSWITYIQYGVYLVVGVIGLRISIYIVQMLLRLRKGYRPVFSSPPSYRQQIHIRRDQELPDGEDREEDGGEKGGNRSWPWQIEYIHFLIRQLIRLLTWLYSNCRDLIYKSFQTLHQLTSAAATATRDFIRTEASYISYGWQYFLEALQAAMQTAGETLASAGGELWATLGRI(SEQ ID NO: 2)Nef Amino Acid Sequence: Encoded by nucleotides 1314-1417 of SEQ ID NO: 1MGGNTSSKPSKRQCRLRERLLQARGENYGQLWEG (SEQ ID NO: 3) 5′LTR/Gag/Pol Nucleotide Sequence: SEQ ID NO: 4 5′LTR: nucleotides 1-827 of SEQ ID NO: 4Gag polypeptide: nucleotides 1046-2560 of SEQ ID NO: 4Env polypeptide (partial): nucleotides 2218-2263 of SEQ ID NO: 4TGGAAGGGATGTTTTACAGTGAGAGGAGGCATAGAATATTAGACACATACTTAGAAAAGGAGGAAGGAATAGTTCCAGATTGGCAGAATTATACACGGGGACCAGGTATTAGATATCCAAAATACTTTGGCTGGCTATGGCAGCTGGAACCAGTGGACGTCTCAGAAGAAAATGATGAGACAAATTGTCTGGTCCATCCAGCGCAGACAAGTCAGTGGGACGACCCATGGGGGGAAACTCTAGTATGGAGATTTAATTCTGCATTGGCTTACACCTATGAGGCTTACATTAGACATCCAGAAGAGTTTGGTTGGAAGTGAGGCCTGTCAGAGGAAGAGGTTAAGCAGAGACTGGCTGACAGGAAGAAGCCAACCACAAAGTAAGATGGCGGACAGAAAGGAAACTAGCTGAGATAGCAGGGACTTTCCAACAAGGGGACGGGCAATGGGTGGAGACTGGGCGGGGGGTATGGGAACGCCCCATTTTACTCTGTATAAATGTACCCGCTTACTGCTCTGTAATCAGTCGCTCTGCGGAGAGGCTGCCAGGTAGAGCCCCGAGTGGATCCCTGGTAGCACTAGCAGGAGAGCCTGGGTGTTCCCTGCTAGACTCTCACTGGTGCTTGGCCAGTACCAGGCAGACGGCTCCACGCTTGCTTGCTTGACTCTCAATAAAGCTGCCATTTAGAAGCAAGTCAGCGTGTGTTCCCATCTCTTCTAGTCGCCGCCTGGTCATTCGGTGTCCTGGCTCGAGGTCTCGGTATCAAGTCCCTGGAACTGTCAGAACCCTCTCACTAAGGGGCAACCCTGAGTGAAAAATCTCTGGCAGTTTGGCGCCCGAACAGGGACATGAGAGACCTGAGAAAGCACACGGCTGAGTGAAGGCAGCAAGGGCGGCAGAAACCAACCGCGACGGAGGAAGACCCGGTGCCAGAGGGCTGAGCGGGACGTGAAGGTAAGAGAGGCCTTCGGGACAGATAGTCCAAAGTTTGTGTAGCTATAGAGCTGTTTCCCTACCCTCAAGGAGGGTAGAAGTATAGCGGGAGATGGGCGCGAGACACTCCGTCTTGTCAGGGAAAAAAGCAGATGAATTAGAAAAAGTTAGGTTACGGCCCGGCGGAAAGAAAAAGTATATGTTAAAGCATATAATATGGGCAGCAAAAGAATTGGACAGATTCGGATTGGCAGAAGACCTGTTGGAAAACAAACAAGGATGTCAAAGAATATTAGAAGTTTTAACCCCATTAATGCCAACAGGCTCAGAAAATTTAAAGAGTTTGTATAATACTGTCTGCGTAGTTTGGTGTTTGCACGCAGAAGAGAAAGTGAAACACACAGAGGAAGCAAAGCAGTTGGTACAGAGACATCTAGTGGCAGAAACTAAAACTGCAGAAAAAATACCAGCAAAAAGTAGACCAACAGCTCCACCTAGTGGAGGAAATTATCCAGTGCAGCAAGTAGGTGGAAATTATGTCCACTTACCATTAAGCCCCAGAACTTTAAATGCCTGGGTAAAATTAGTAGAGGAAAAGAAATTTGGAGCAGAAGTAGTGCCAGGCTTTCAGGCACTGTCAGAAGGCTGCACACCTTATGATATTAATCAGATGCTAAATTGTGTAGGGGAACATCAAGCGGCTATGCAAATAATTAGAGAAATTATCAATGAGGAAGCAGCAGACTGGGACGCACAGCATCCAAGGCAGCTACCGGCACCTCCGGGGCTGCGCGACCCGTCAGGGTCAGATATAGCAGGAACCACCAGTACTGTAGAAGAGCAAATAGAGTGGATGTATAGACAAGGAAATCCTGTCCCAGTAGGACAAATTTACAGGAGATGGATTCAGCTAGGATTACAAAAATGTGTAAGAATGTACAATCCCACTAACATTCTAGACGTAAAGCAAGGTCCAAAAGAGCCATTCCAAGTTTATGTAGACAGGTTCTACAAAAGTTTGAGAGCAGAACAAACAGACCCAGCAGTGAAGAATTGGATGACCCAAACACTGCTGATCCAAAATGCCAACCCTGATTGCAAACTAGTATTAAAAGGATTGGGAATGAATCCCACCTTAGAAGAAATGTTAACAGCTTGTCAGGGAGTGGGAGGTCCTGGACAAAAGGCTAGGTTAATGGCCGAGGCAATGAAGGAAGCCTTTAATGGCTCCTTCGCGGCCGTGCAGATGAGAGGGAAACAACAGAAGGGGGCATCAACTATTAGATGCTTTAATTGTGGGAAACCAGGCCACACTGCCAGAAATTGCAGGGCACCAAGAAGAAAGGGGTGCTGGAAATGTGGAGAGGAAGGACACATGCAAGCAAACTGCCCAAACCAACGGGCGGGTTTTTTAGGGTTAGGACCATGGGGAAAGAAGCCTCGCAACTTCCCCATGAGACAGATGCCAGAGGGACTGACCCCATCAGCCCCTCCGGACCCAGCAGCAGAAATGCTCGAGGAGTATATGCAGAAGGGGAAAAGTCAGAGGGAGCAGAGGGAGAGACCTTACAAAGAGGTGACGGAGGACTTGCTGCACCTCAGTTCTCTCTTTGGAAAAGACCAGTAGTCACAGCATATATAGAGGATCAGCCAGTACAGGTACTGCTAGATACAGGAGCTGATGACTCTATAGTGGCAGGGATAGAATTAGGACTTAATTACAAGCCAAA (SEQ ID NO: 4 ) Gag Amino Acid Sequence: SEQ ID NO: 5MGARHSVLSGKKADELEKVRLRPGGKKKYMLKHIIWAAKELDRFGLAEDLLENKQGCQRILEVLTPLMPTGSENLKSLYNTVCVVWCLHAEEKVKHTEEAKQLVQRHLVAETKTAEKIPAKSRPTAPPSGGNYPVQQVGGNYVHLPLSPRTLNAWVKLVEEKKFGAEVVPGFQALSEGCTPYDINQMLNCVGEHQAAMQIIREIINEEAADWDAQHPRQLPAPPGLRDPSGSDIAGTTSTVEEQIEWMYRQGNPVPVGQIYRRWIQLGLQKCVRMYNPTNILDVKQGPKEPFQVYVDRFYKSLRAEQTDPAVKNWMTQTLLIQNANPDCKLVLKGLGMNPTLEEMLTACQGVGGPGQKARLMAEAMKEAFNGSFAAVQMRGKQQKGASTIRCFNCGKPGHTARNCRAPRRKGCWKCGEEGHMQANCPNQRAGFLGLGPWGKKPRNFPMRQMPEGLTPSAPPDPAAEMLEEYMQKGKSQREQRERPYREVTEDLLHLSSLFGKDQ (SEQ ID NO: 5) Pol Amino Acid Sequenc: SEQ ID NO: 6MWETRPHCQKLQGTKKKGVLEMWRGRTHASKLPKPTGGFFRVRTMGKEASQLPHETDARGTDPISPSGPSSRNARGVYAEGEKSEGAEGETLQRGDGGLAAPQFSLWKRPVVTAYIEDQPVQVLLDTGADDSIVAGIELGLNYKP (SEQ ID NO: 6)

In some embodiments, the invention provides a nucleic acid thathybridizes under stringent conditions to the nucleotide sequence of SEQID NO:1 or SEQ ID NO:4. In certain embodiments, stringent conditionsinclude, but are not limited to (1) wash in aqueous prehybridizationbuffer (6×SSC, 5×Denhardt's reagent, 0.5% SDS) at 68° C.; (2)hybridization of the probe in aqueous hybridization buffer (6×SSC,5×Denhardt's reagent, 0.5% SDS, 1 μg/ml poly(A), 100 μg/ml salmon spermDNA) at 68° C.; and (3) wash in 2×SSC, 0.5% SDS at room temperature.Alternatively, stringent conditions include, but are not limited to (1)wash in formamide prehybridization buffer (6×SSC, 5×Denhardt's reagent,0.5% SDS, 50% formamide) at 42° C.; (2) hybridization of the probe informamide hybridization buffer (6×SSC, 5×Denhardt's reagent, 0.5% SDS,50% formamide, 1 μg/ml poly(A), 100 μg/ml salmon sperm DNA) at 42° C.;and (3) wash in 2×SSC, 0.5% SDS at room temperature. Additionalvariations on these stringent conditions are know to one of skill theart and may be found, for example, in Chapter 6 of Sambrook & Russell,Molecular Cloning: A Laboratory Manual, CSHL Press, 2001, which isincorporated by reference herein. In exemplary embodiments, theinvention provides a nucleic acid that is at least about 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQID NO:1 or SEQ ID NO:4, or fragments thereof.

In some embodiments, the nucleic acid that hybridizes under stringentconditions is a probe or a primer. The probe or the primer maybe used todetect the presence of HIV-2NWK08F or other HIV strains in a biologicalsample. Exemplary primers are set forth below in Tables 2 and 3.

TABLE 2  HIV-2NWK08F Env-Nef Primers PCR Fragment Primer Primer SequenceSize 1 Outer Forward 5′-GGA GGA GGA GAT CCG GAA GT-3′ SEQ ID NO: 7492 bp Outer Reverse 5′-AGA ACC TGC CGT TGC GAG AA-3′ SEQ ID NO: 8 2Outer Forward 5′-TCC ACA GTG ACC AGT CTC AT-3′ SEQ ID NO: 9 333 bpOuter Reverse 5′-GAT GGC AGT GAC GCG TGT CT-3′ SEQ ID NO: 10Inner Forward 5′-TGA GTG CAG AGG TGG CAG AA-3′ SEQ ID NO: 11Inner Reverse 5′-GTG ACG CGT GTC TGG AGG TT-3′ SEQ ID NO: 12 3Outer Forward 5′-GCA GGC ACA GAT TCA ACA AG-3′ SEQ ID NO: 13 381 bpInner Reverse 5′-GCA ACT GCT GAA TAG CCA AGT C-3′ SEQ ID NO: 14 4Outer Forward 5′-GGC TGG GAT AGT GCA GCA ACA GCA ACA G-3′ SEQ ID NO: 15485 bp Outer Reverse 5′-AAG CGG GAG GGG AAG AGA ACA CTG GCC-3′SEQ ID NO: 16 Inner Forward 5′-TGT TGG ACG TGG TCA AGA GAC AAC-3′SEQ ID NO: 17 Inner Reverse 5′-GGG AGG GGA AGA GAA CAC TGG CCT ATA-3′SEQ ID NO: 18 5 Outer Forward 5′-GAG AAG AAG ACG GTG GAG AA-3′SEQ ID NO: 19 341 bp Inner Reverse 5′-GGA TTG CGA GTA TCC ATC TTC C-3′SEQ ID NO: 20

TABLE 3  HIV-2NWK08F LTR-Gag Primers PCR  Fragment PrimerPrimer Sequence Size 1 Outer Forward 5′-AGA AGG CTA GCC GCA AGA GG-3′SEQ ID NO: 21 511 bp Outer Reverse 5′-TAC CTT CAC GTC CCG CTC AG-3′SEQ ID NO: 22 Inner Forward 5′-GAC ACA GCA GGG ACT TTC CA-3′SEQ ID NO: 23 Inner Reverse 5′-TTC CTC CGT CGC GGT TGG TT-3′SEQ ID NO: 24 2 Outer Forward 5′-ACT CCT GAG TAC GGC TGA GT-3′SEQ ID NO: 25 318 bp Outer Reverse 5′-CAA CAG GTC TTC TGC CAA TC-3′SEQ ID NO: 26 Inner Forward 5′-GGC TGA GTG AAG GCA GTA AG-3′SEQ ID NO: 27 Inner Reverse 5′-TCT GCC AAT CCG AAT CTG TC-3′SEQ ID NO: 28 3 Outer Forward 5′-TGG GAG ATG GGC GCG AGA AAC TCC GTC-3′809 bp SEQ ID NO: 29 Outer Reverse5′-TCC ACA TTT CCA GCA GCC CTG TCT TCT-3′ SEQ ID NO: 30 Inner Forward5′-AGG GAA GAA AGC AGA TGA ATT AGA A-3′ SEQ ID NO: 31 Inner Reverse5′-GCA TTT TGA ATC AGC AGT GTT TGA GTC ATC CA-3′ SEQ ID NO: 32 4Outer Forward 5′-ACG CAC AGC ATC CAA G-3′ SEQ ID NO: 33 545 bpOuter Reverse 5′-CTT GAG CCA TGG GGA AAT TG-3′ SEQ ID NO: 34Inner Forward 5′-GGA GAT GGA TTC AGC TAG GA-3′ SEQ ID NO: 35Inner Reverse 5′-GGG GCT TCT TTC CCC ATG GAC C-3′ SEQ ID NO: 36

In exemplary embodiments, the nucleic acid comprises at least sixnucleic acids derived from the HIV-2NWK08F genome. In particularembodiments, the nucleic acid comprises at least 6, 8, 10, 12, 15, 20,25, 30, 40, or 50 consecutive nucleotides derived from the HIV-2NWK08Fgenome. In some embodiments, the nucleic acid comprises at least 6, 8,10, 12, 15, 20, 25, 30, 40, or 50 consecutive nucleotides of SEQ ID NO:1or SEQ ID NO:4. In some embodiments, the nucleic acid is no longer thanabout 50, 75, 100, or 200 nucleotides. In another embodiment, thenucleic acids of the invention encode a viral protein or fragmentthereof, wherein the viral protein or fragment possesses the biologicalactivity associated with the protein.

In some embodiments, the nucleic acids of the invention are attached toa detectable label. Examples of detectable labels include, but are notlimited to, chromogens, radioisotopes, chemiluminescent compounds,visible or fluorescent particles, and enzymes. In the case of enzymeslabels (e.g., alkaline phosphatase or horseradish peroxidase), additionof a chromo-, fluoro-, or lumogenic substrate results in the generationof a detectable signal.

The nucleic acids of the invention may be used to differentially detectHIV-2NWK08F from other subtype F HIV-2 viruses or from other HIV-1,HIV-2, or SIV strains. Alternatively, the nucleic acids of the inventionmay be used to detect any HIV or SIV strain. One of skill in the artwould understand how to design primers and probes so as topreferentially detect HW-2NWK08F or to detect all HIV and SIV strains.Briefly, one could examine the sequences of a number of HIV and SIVstrains and select regions of high or low homology. If the primers andprobes are to preferentially detect HIV-2NWK08F, they are derived fromregions of the genome with low homology to other strains and subtypes.Examples of regions with low homology include regions encoding the Envpolypeptide. If the primers and probes are to detect all or some otherHIV or SIV strains, they are derived from regions of the genome withhigh homology to other strains and subtypes. Examples of regions withhigh homology between strains include regions encoding the viral reversetranscriptase.

As used herein, a nucleic acid with a designated sequence or “derivedfrom” a designated source refers to a nucleotide sequence that ishomologous (i.e., identical) to or complementary to the designatedsequence or source, or a portion thereof.

The nucleic acids of the invention may be produced by any method knownto one of skill in the art. In exemplary embodiments, the nucleic acidsare isolated from cells infected with HIV-2NWK08F.

Methods for isolating viral RNA or DNA are well known in the art. Anexemplary method for extracting viral RNA or DNA from infected cells isdescribed in Laure et al., Lancet 2(8610):538-41 (1988), the contents ofwhich are incorporated by reference herein. Because infected cellscontain copies of reverse transcribed viral DNA while whole virionscontain only viral DNA, the methods for extracting the two nucleic acidsdiffer slightly. To extract viral DNA, infected cells are separated fromother blood constituents by centrifugation in a gradient, such as, e.g.,a Ficoll gradient. The cells are lysed in a appropriate lysis buffer(e.g., 10 mM Tris pH 8, 10 mM EDTA, 10 mM NaCl, 0.5% SDS, 100 μg/mgproteinase K) for approximately 2 hours at 60° C. The DNA is thenextracted with phenol and precipitated with ethanol. To isolate viralRNA, the same procedure is carried out on the serum, plasma, or bloodleukocytes of infected patients or on the supernatant of infected cellcultures. Once extracted, the RNA may be transformed from singlestranded RNA to double stranded DNA by performing a reversetranscriptase reaction, such as that described in U.S. Pat. No.6,020,123, incorporated by reference herein.

The nucleic acids of the invention may also be produced recombinantly.Briefly, the nucleic acid is inserted into a vector and amplified in ahost organism. Suitable vectors can be chosen or constructed, containingappropriate regulatory sequences. Vectors may be plasmids or viral,e.g., phage or phagemid, as appropriate. Many known techniques andprotocols for manipulation of nucleic acid in preparation of nucleicacid constructs, mutagenesis, sequencing, introduction of DNA and/orvectors into cells, gene expression, and analysis of proteins aredescribed in detail in Sambrook & Russell, Molecular Cloning: ALaboratory Manual, CSHL Press, 2001, the contents of which areincorporated by reference herein.

Alternatively, the nucleic acid may be amplified via the polymerasechain reaction using any of the exemplary primers disclosed herein orother primers derived from the nucleotide sequence of HIV-2NWK08F oranother HIV virus (e.g., a different HIV-2 subtype F virus, a differentHIV-2 subtype virus, or an HIV-1 or SIV virus). Methods for practicingnucleic acid amplification are described in Sambrook & Russell,Molecular Cloning: A Laboratory Manual, CSHL Press, 2001. Finally, thenucleic acids of the invention may be chemically synthesized using wellknow methods available from numerous commercial vendors, including, bynot limited to, Applied Biosystems and Integrated DNA Technologies.Methods for purifying and handling chemically synthesized nucleic acidsare described in Sambrook & Russell, Molecular Cloning: A LaboratoryManual, CSHL Press, 2001.

Polypeptides

The invention also provides amino acid sequences of HIV-2NWK08Fpolypeptides. The polypeptides of the invention are useful for detectingthe presence of HIV-2NWK08F-specific antibodies in patient serum. Thepolypeptides of the invention are also useful for generatingHIV-2NWK08F-specific antibodies. The polypeptides may be naturallyoccurring, recombinantly produced, or synthetic.

In exemplary embodiments, the polypeptides of the invention comprise atleast six consecutive amino acids derived from a polypeptide encoded bythe HIV-2NWK08F genome. In particular embodiments, the polypeptidecomprises at least 6, 8, 6, 8, 10, 12, 15, 20, 25, 30, 40, or 50consecutive amino acids derived from HIV-2NWK08F. In some embodiments,the polypeptide comprises at least 6, 8, 6, 8, 10, 12, 15, 20, 25, 30,40, or 50 consecutive amino acids of SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:5, or SEQ ID NO:6. In some embodiments, the polypeptide is no longerthan about 10, 11, 12, 13, 14, 15, 25, 50, 75, 100, 200, 300, 400, or1000 amino acids. In other embodiments, the polypeptides comprises anentire viral protein, or a fragment thereof, wherein the viral proteinor fragment possesses the biological activity associated with thatprotein.

An amino acid sequence derived from a designated polypeptide or sourcemeans that the amino acid sequence is homologous (i.e., identical) tothe sequence of the designated polypeptide, or a portion thereof. Anamino acid sequence from a designated nucleic acid sequence refers to apolypeptide having an amino acid sequence identical to that of apolypeptide encoded in the sequence, or a portion thereof.

The term “polypeptide” refers to a polymer of amino acids and does notrefer to a specific length; thus, peptides, oligopeptides, and proteinsare included within the definition of polypeptide. This term does notexclude post-expression modifications of the polypeptide, for example,glycosylation, acetylation, phosphorylation, pegylation, addition of alipid moiety, or the addition of any organic or inorganic molecule.Included within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids) and polypeptides with substituted linkages, as well asother modifications known in the art, both naturally occurring andnon-naturally occurring.

In some embodiments, the polypeptides of the invention are derived fromHIV-2NWK08F Gag or Env polypeptides or fragments thereof. In someembodiments, the polypeptides of the invention are immunogenic. Inparticular embodiments, the polypeptides of the invention comprise SEQID NO:2, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:6, or fragments thereof.

The polypeptides of the invention may be prepared by any method known toone of skill in the art, including those described in Sambrook &Russell, Molecular Cloning: A Laboratory Manual, CSHL Press, 2001, thecontents of which are incorporated by reference herein. The polypeptidesmay be recombinantly expressed and purified. For example, DNA encodingthe desired polypeptide may be ligated into an expression vector for anyconvenient host, either eukaryotic or prokaryotic. The polypeptide isexpressed in a culture of the host cells and then isolated from lysedcells or the culture medium and purified.

Alternatively, the polypeptides of the invention may be isolated frominfected cell cultures and purified. The purification of thepolypeptides may be performed by techniques known in the art, such as,e.g., differential extraction, salt fractionation, cetrifugation, andion exchange, affinity, or size exclusion chromatography.

Polypeptides of the invention may also be produced by chemicalsynthesis. For example, the polypeptides may be prepared withsolid-phase synthesis such as that described in Bodansky et al., ThePractice of Peptide Synthesis, Springer-Verlag, 1994.

Antibodies

The invention also provides HIV-2NWK08F-specific antibodies. Theantibodies may be generated against the entire virion or against aspecific polypeptide, including both whole proteins and peptidefragments. The antibodies of the invention are useful for the detectionof HIV-2NWK08F infection in patients or cell culture, for the detectionof HIV-2NWK08F antigens or virions, and for the treatment of patientsinfected with HIV-2NWK08F or similar viruses. The antibodies of theinvention may also be used as therapeutics for the treatment of HIVinfection. In a non-limiting example, antibodies specific for theHIV-2NWK08F Env protein may be used to prevent the binding of the Envprotein to cells.

The term “antibody” as used herein includes polyclonal antibodies,monoclonal antibodies, antibody compositions with polyepitopespecificities, bispecific antibodies, diabodies, humanized antibodies,and other purified preparations of antibodies and recombinantantibodies. The antibodies can be whole antibodies of any isotype, e.g.,IgG, IgA, IgE, IgM, etc, or fragments thereof, which bind the antigen ofinterest. In a specific example of an antibody used in the presentinvention, the antibody is an IgG antibody. Antibodies can be fragmentedusing conventional or other techniques and the fragments screened forbinding to an antigen of interest. Generally, an antibody fragmentcomprises the antigen-binding and/or the variable region of an intactantibody.

The term “antibody fragment” includes segments of proteolyticallycleaved or recombinantly prepared portions of an antibody molecule thatcan selectively bind to a selected protein. Non-limiting examples ofsuch proteolytic and/or recombinant fragments include Fab, F(abγ)₂,Fabγ, Fv, and single chain antibodies (scFv) containing a V[L] and/orV[H] domain joined by a peptide linker, domain antibodies (dAbs),Nanobodies® (antibody-derived biological therapeutic agents that containthe unique structural and functional properties of naturally-occurringheavy-chain antibodies), and UniBodies (antibodies lacking the hingeregion). The scFvs may be covalently or noncovalently linked to formantibodies having two or more binding sites.

The term “antigen” refers to any part of a virus that an antibody mayspecifically bind to. This includes polypeptides and modifying groups,such as glycoproteins, that may be attached to the polypeptides. It mayinclude the whole virus, part of a virus, a whole protein, or part of aprotein. An antibody may only bind to a part of the antigen.

The term “epitope” refers to a portion of an antigen that is responsiblefor specific interactions with the antibody. An epitope may be a peptideof at least about 6, 8, 10, 11, 12, 13, or 15 amino acids in length.

Antibodies specific for polypeptides of the invention can be prepared byany technique known to one of skill in the art. For example, theantibodies of the invention can be prepared via traditional hybridomatechniques (Kohler and Milstein, Nature 256:495-499 (1975), incorporatedby reference herein), recombinant DNA methods (U.S. Pat. No. 4,816,567,incorporated by reference herein), or phage display techniques usingantibody libraries (Clackson et al., Nature 352:624-628 (1991); Marks etal., J Mol Biol 222:581-597 (1991), both of which are incorporated byreference herein). For various other antibody production techniques, seeHarlow et al., Antibodies: A Laboratory Manual, CSHL Press, 1988, thecontents of which are incorporated by reference herein.

Viral Detection Assays

The nucleic acids, polypeptides, and antibodies of the invention areuseful for the detection of the presence of HIV-2 in a biologicalsample. These assays may be used to detect the presence of HIV infectionin a patient or to determine whether cultured cells have been infectedwith the virus. The presence of HIV-2NWK08F nucleic acids, polypeptides,or antibodies in a patient or in a cell culture is indicative of HIVinfection. Due to the homology between different subtypes and strains ofHIV, detection of HIV-2NWK08F nucleic acids, polypeptides, andantibodies may also be used to detect other HIV subtypes and strains.Alternatively, detection of HIV-2NWK08F nucleic acids, polypeptides, orantibodies in a patient or cell culture may be used to determine thatthe patient or cells are infected with HIV-2NWK08F, and not a differentstrain or subtype of HIV.

All of the viral detection assays of the invention involve a step ofproviding a biological sample suspected of being infected with HIV or ofcontaining HIV nucleic acids, polypeptides or anti-HIV antibodies.Accordingly, the first step in the viral detection assays is theidentification of an appropriate patient, mammal, or infected cellculture and providing a biological sample from that source.

“Biological sample” refers to a sample of tissue or fluid isolated froman individual, including but not limited to, for example, whole bloodand components thereof, dried blood, plasma, serum, spinal fluid, lymphfluid, the external sections of the skin, respiratory, intestinal, andgenitourinary tracts, tears, saliva, urine, milk, blood cells, tumors,organs, and also samples of in vitro cell culture constituents,including, but not limited to, putatively virally infected cells, cellsputatively expressing viral polypeptides, cell components, conditionedmedium resulting from the growth of cells in cell culture medium, andcell free supernatant of one of the tissues or fluids listed above.

Nucleic Acid-Based Assays

Nucleic acid probes and primers are useful in identification of thevirus, further characterization of the viral genome, and detection ofthe virus in diseased individuals. Nucleic acid-based assays are apreferred method for measuring HIV viral load in infected patients. Innucleic acid-based assays, an essential step is the provision of boththe detecting nucleic acid, such as a primer or a probe and the nucleicacid to be detected, such as viral RNA or DNA. Methods for preparingboth the detecting nucleic acid and the nucleic acid to be detected areprovided herein.

Methods for selecting probes and primers and determining the specificityof these nucleic acids are known to those of skill in the art. Thesequence and length of the probe or primer will depend on the proposeduse. If the probe or primer is to be used to differentially detect thepresence of HIV-2NWK08F, the sequence of the probe or primer will bespecific to this virus, i.e., derived from an area of the viral genomethat is not conserved between virus subtypes or strains. Viral genesthat show low levels of homology between subtypes and strains includethe envelope gene. If the probe or primer is to be used to detect allHIV-2 viruses or all HIV viruses in general, it will be derived from anarea of the viral genome that is conserved between virus subtypes orstrains. Viral genes that show high levels of homology between subtypesand strains include certain regulatory proteins, such as, e.g., thegenes encoding the viral reverse transcriptase.

For diagnostic use of probes, the biological sample to be analyzed, suchas blood or serum, may be treated, if desired, to extract the nucleicacids contained therein. The resulting nucleic acid from the sample maybe subjected to gel electrophoresis or other size separation techniques;alternatively, the nucleic acid sample may be dot blotted without sizeseparation. The probes are then labeled. Suitable labels, and methodsfor labeling probes are known in the art, and include, for example,radioactive labels incorporated by nick translation or kinasing, biotin,fluorescent probes, and chemiluminescent probes. The nucleic acidsextracted from the sample are then treated with the labeled probe underhybridization conditions of suitable stringencies. Usually highstringency conditions are desirable in order to prevent false positives.The stringency of hybridization is determined by a number of factorsduring hybridization and during the washing procedure, includingtemperature, ionic strength, length of time, and concentration offormamide. These factors are outlined in, for example, Sambrook &Russell, Molecular Cloning: A Laboratory Manual, CSHL Press, 2001.

If the HIV-2NWK08F genome sequences are present in serum of infectedindividuals at relatively low levels, detection may require thatamplification techniques be used in hybridization assays. Suchtechniques are known in the art. A particularly desirable technique mayfirst involve amplification of the target HIV-2NWK08F sequences inserum, plasma, or cells approximately 10,000-fold, i.e., toapproximately 10⁶ sequences/ml. This may be accomplished, for example,by polymerase chain reactions (PCR). The amplified sequences may then bedetected using a hybridization assay. These hybridization assays, whichshould detect sequences at the level of 10⁶ copies/ml, utilize nucleicacid multimers which bind to single-stranded analyte nucleic acid, andwhich also bind to a multiplicity of single-stranded labeledoligonucleotides. A suitable solution phase sandwich assay may be usedwith labeled polynucleotide probes.

The probes and primers of the invention may be used to quantitate viralnucleic acid levels by real-time PCR. Real-time PCR refers to apolymerase chain reaction that is monitored, usually by fluorescence,over time during the amplification process, to measure a parameterrelated to the extent of amplification of a particular sequence, such asthe extent of hybridization of a probe to amplified target sequences.The initial nucleic acids of the reaction are synthesized from the RNAtemplate using reverse transcriptase. Then the DNA generated within aPCR reaction is detected on a cycle by cycle basis during the PCRreaction. The amount of DNA increases with the amount of templatesequences present in the original sample. When enough amplificationproducts are made, a threshold is reached at which the PCR products aredetected.

The HIV-2NWK08F probes and primers are a length that allows thedetection of unique viral sequences by hybridization. These nucleicacids can be prepared using routine methods, including automatedoligonucleotide synthetic methods. The nucleic acids may be a complementto any unique portion of the HIV-2NWK08F genome. For use as probes,complete complementarity is desirable, though it may be unnecessary asthe length of the fragment is increased. Primers used in the methods ofthe invention include oligonucleotides of sufficient length andappropriate sequence to provide specific initiation of polymerization ofa HIV or HIV-2NWK08F nucleic acid in a polymerase chain reaction (PCR).Conditions and reagents for performing nucleic acid amplificationutilizing PCR and other nucleic acid amplification techniques are knownto one of skill in the art and may be found in, e.g., Sambrook &Russell, Molecular Cloning: A Laboratory Manual, CSHL Press, 2001. Thenucleic acids that make up the probes or primers of the invention may beat least about, e.g., 6, 8, 10, 12, 15, 20, 25, 30, 40, or 50nucleotides in length. The nucleic acids may have a maximum length ofabout, e.g., 50, 75, 100, or 200 nucleotides.

The probes and primers of the invention can be packaged into diagnostickits. Diagnostic kits include the probe and/or primer nucleic acids,which may be labeled; alternatively, the probe or primer nucleic acidsmay be unlabeled and the ingredients for labeling may be included in thekit in separate containers. The kit may also contain other suitablypackaged reagents and materials needed for the particular amplificationand/or hybridization protocol, for example, standards, enzymes,nucleotide triphosphates, wash buffers, as well as instructions forconducting the test.

Immunoassays

Immunoassays may be used to detect anti-HIV-2NWK08F antibodies inpatient serum or to detect HIV-2NWK08F-specific antigens in patientserum. Immunoassays can be used to test for the presence of HIVantibodies or antigens in blood, oral mucosal transudate (OMT) fluid,saliva, and urine, or cell-free supernatants of these fluids.Immunoassays are currently more commonly used for diagnosing HIVinfection than nucleic acid assays.

HIV immunoassays include, but are not limited to, Western blot assaysand enzyme-linked immunosorbent assays (ELISA). An immunoassay for viralantigen may use, for example, a monoclonal antibody directed towards aviral epitope, a combination of monoclonal antibodies directed towardsepitopes of one viral polypeptide, monoclonal antibodies directedtowards epitopes of different viral polypeptides, polyclonal antibodiesdirected towards the same viral antigen, polyclonal antibodies directedtowards different viral antigens, or a combination of monoclonal andpolyclonal antibodies.

An immunoassay for viral antibodies may use, for example, a HIV-2NWK08Fviral antigen, such as, e.g., fragments of the Env, Pol, Gag, or Nefpolypeptides.

Immunoassay protocols may be based, for example, upon competition, ordirect reaction, or sandwich type assays. Protocols may also, forexample, use solid supports, or may be by immunoprecipitation. Mostassays involve the use of labeled antibody or polypeptide. The labelsmay be, for example, fluorescent, chemiluminescent, radioactive, or dyemolecules. Assays which amplify the signals from the probe are alsoknown. Examples of which are assays which utilize biotin and avidin, andenzyme-labeled and mediated immunoassays, such as ELISA assays.

Typically, an immunoassay for anti-HIV-2NWK08F antibody will involveselecting and preparing the test sample, such as a biological sample,and then incubating it with an antigenic (i.e., epitope-containing)HW-2NWK08F polypeptide under conditions that allow antigen-antibodycomplexes to form. Such conditions are well known in the art. Thepolypeptide may be bound to a solid support. Examples of solid supportsthat can be used are nitrocellulose in membrane or microtiter well form,polyvinylchloride in sheets or microtiter wells, polystyrene latex, inbeads or microtiter plates, polyvinylidine fluoride (known asImmobilon™), diazotized paper, nylon membranes, activated beads, andProtein A beads. In certain embodiments, these assays are performedusing either Immulon™ microtiter plates (Dynatech) or specular-finished0.25-inch polystyrene beads (available from Precision Plastic Ball). Thesolid support is typically washed after separating it from the testsample.

In another embodiment, the test sample is incubated with antigen insolution under conditions that will precipitate any antigen-antibodycomplexes that are formed, as is known in the art. The precipitatedcomplexes are then separated from the test sample by, for example,centrifugation. The complexes formed comprising anti-HIV-2NWK08Fantibody are then detected by any of a number of techniques. Dependingon the type of assay, the complexes can be detected with labeledanti-xenogeneic Ig or by measuring the amount of bound, labeledcompeting antibody.

In immunoassays where HIV-2NWK08F polypeptides are the analyte, the testsample, typically a biological sample, is incubated withanti-HIV-2NWK08F antibodies again under conditions that allow theformation of antigen-antibody complexes. Various formats can beemployed, such as the “sandwich” assay. In this assay, antibody is boundto a solid support, incubated with a test sample, washed, incubated witha labeled anti-analyte antibody, and washed again. Analyte is detectedby determining if the second antibody is bound to the support. In acompetitive format, which can be either heterogeneous or homogeneous, atest sample is usually incubated with and antibody and a labeled,competing antigen either sequentially or simultaneously. These and otherformats are well known in the art.

Treatment of Infected Patients

This invention also provides methods of treating patients infected withHIV-2NWK08F. Methods of treating HIV infections are known in the art.Patients infected with HIV-2NWK08F can be treated by any known anti-HIVtherapeutic, such as those described in Panel on AntiretroviralGuidelines for Adults and Adolescents. Guidelines for the use ofantiretroviral agents in HIV-1-infected adults and adolescents.Department of Health and Human Services. Jan. 29, 2008; 1-128,incorporated by reference herein. The invention also provides methods oftreating opportunistic infections that accompany the immunosuppressioncaused by HIV infection. These opportunistic diseases and theirtreatments are described in Panel on Guidelines for the Prevention andTreatment of Opportunistic Infections in HIV-Infected Adults andAdolescents. Guidelines for Prevention and Treatment of OpportunisticInfections in Adults and Adolescents. Department of Health and HumanServices. Jun. 20, 2008; 1-302, incorporated by reference herein.

In addition, the HIV-2NWK08F virus, nucleic acids, and proteins of theinvention may be used to develop new therapeutic antibodies and vaccinesfor the treatment of HIV-2 infections. The vaccines may be derived fromthe whole virus, the virion, viral proteins, or viral nucleic acids.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

EXAMPLES Example 1 Identification of Infected Patient

Patient X, a 68 year old man from Sierra Leone, tested positive forantibodies to HIV in a test that detects the presence of HIV-1 or HIV-2antibodies. Subsequent testing for HIV-1 antibodies in a Western blotshowed that the patient was not infected with HIV-1. His HIV-1 viralload was tested in an Real-time Polymerase Chain Reaction (RT-PCR) assay(LabCorp) and was below the lower limit of the assay. HIV-2 viral DNAtesting performed at two commercial laboratories (Viromed; FocusTechnologies) was negative, as was an HIV-2 Western blot. HIV-2 viralDNA was finally detected with the use of primers derived from HIV-2subtype F (as described in Example 3).

Example 2 Isolation and Propagation of HIV

Normal donor PBMCs were stimulated with PHA at 5 μg/ml for three daysand then re-suspended in RPMI supplemented with 10% fetal bovine serumand 20 units/ml of IL-2. To isolate the virus, these stimulated normaldonor PBMCs were then co-cultured with PBMCs from Patient X. The culturewas split every 3-4 days and supernatant was collected for p27measurement by EIA for SW p27 (Zeptometrix), which detected the p27protein of HIV-2NWK08F. PBMCs were collected at each time point andtheir DNA was extracted.

This co-culturing process was performed with three different populationsof normal donor PBMCs. HW-2NWK08F virus replicated each time, with thepeak p27 concentration exceeding 10 μg/ml in each co-culture (testedwith Zeptometrix SW p27 kit).

Example 3 Amplification and Sequencing of Viral RNA

Portions of the gag and env genes were amplified as described in Chen etal., J Virol 71:3953-3960 (1997). Briefly, DNA was extracted from eachPBMC culture on day 13 using a DNA extraction kit (Qiagen, Valencia,Calif.). Nested primers were used to amplify a 438-bp env fragment.Traditional polymerase chain reaction (PCR) was performed in a Tpersonalthermocycler (Biometra, Hannover, Germany) using a PCR Master Mix Kit(Applied Biosystems, Foster City, Calif.). These reactions wereperformed in a 50 μl volume which contained approximately 350 ng of DNAand 20 pmol of each primer. The first round of PCR had an initialactivation step at 95° C. for 5 minutes followed by 30 cycles of 95° C.for 20 seconds, 45° C. for 1.5 minutes, and 72° C. for 2 minutes withenv outer primer pair EF-1 and ER-1. The second round of PCR wasperformed using inner primers EF-2 and ER-2. This round consisted of 30cycles of 94° C. for 20 seconds, 55° C. for 1.5 minutes, and 72° C. for2 minutes. Additionally, both rounds of PCR consisted of an extensionstep at 72° C. for 8 minutes.

(SEQ ID NO: 37) EF-1: GGCTGGGATAGTGCAGCAACAGCAACAG (SEQ ID NO: 38)ER-1: GGGAGGGGAAGAGAACACTGGCCTATA (SEQ ID NO: 39)EF-2: TGTTGGACGTGGTCAAGAGACAAC (SEQ ID NO: 40)ER-2: AAGCGGGAGGGGAAGAGAACACTGGCC

The same procedure was used to amplify a 826 bp gag fragment using thefollowing primers:

(SEQ ID NO: 41) GF-1: TGGGAGATGGGCGCGAGAAACTCCGTC (SEQ ID NO: 42)GR-1: TCCACATTTCCAGCAGCCCTGTCTTCT (SEQ ID NO: 43)GF-2: AGGGAAGAAAGCAGATGAATTAGAA (SEQ ID NO: 44)GR-2: GCATTTTGAATCAGCAGTGTTTGAGTCATCCA

The gag and env PCR products were cloned and sequenced as described inChen et al., J Virol 70:3617-27 (1996).

Example 4 Phylogenetic Analysis

Gag and Env nucleotide sequence alignments were obtained from the LosAlamos National Laboratory HIV Sequence Database Newly derivedHIV-2NWK08F sequences were aligned using the CLUSTAL W profile alignmentoption. The resulting alignments were adjusted manually when necessary.Regions of ambiguous alignment and all gap-containing sites wereexcluded.

Phylogenetic trees were inferred from the nucleotide sequence alignmentsby the neighbor-joining method, using the HKY85 model of nucleotidesubstitution implemented in PAUP*. The reliability of the branchingorder was assessed by performing 1,000 bootstrap replicates, again usingneighbor joining and the HKY85 model. Phylogenetic trees were alsoinferred by the maximum likelihood method, using PAUP* with modelsinferred from the alignment by the use of Modeltest (Posada, et al.,Syst Biol 50:580601 (2001); Swofford, PAUP*. Phylogenetic analysis usingparsimony (*and other methods), version 4. Sinauer Associates,Sunderland, Mass. (1999)). These trees are shown in FIGS. 1 and 2.

Example 5 Detection of Anti-HIV Antibodies in Patient Serum

A sample of Patient X's blood was sent to LabCorp® of Burlington, N.C.for detection of HIV antibodies. The assay used to assess the presenceof HIV-1 antibodies was Protocol Number 005462, a Western blottingprocedure that detects antibodies to Gp41, Gp120, Gp160, p18, p24, p31,p40, p51, p55, and p64. This Western blot for HIV-1 antibodies wasnegative. HIV-2 antibodies were positively detected using ProtocolNumber 163550, an Enzyme Immunoassay (EIA) that differentially detectsantibodies to HIV-2, but not HIV-1.

Example 6 Real Time Reverse Transcription-PCR for HIV-2 Proviral Load

Proviral DNA from PBMCs was isolated using a DNA extraction kit fromQIAGEN®. The DNA quality was confirmed with a spectrophotometer. 48 ngand 480 ng of DNA was subsequently quantified with real time PCR usingthe following primers and probe derived from the HIV-2NWK08F env gene.

(SEQ ID NO: 45) Forward Primer AAGAATTGTTGCGATTGACAGTCT (SEQ ID NO: 46)Reverse Primer TGCACACCCCCATGAATTTA (SEQ ID NO: 47) ProbeACTAAAAACCTCCAGACACGCGTCACTGC

The PCR was performed in TaqMan Universal PCR Master Mix on an AppliedBiosystems 7300 Real time PCR System (Applied Biosystems, Foster City,Calif.). The real time PCR conditions were as described in Table 4.

TABLE 4 Temperature Stage (° C.) Time (min:secs) Repeat 1 50 2:00 1 2 9510:00  1 3 95 0:15 40 60 1:00

The real time PCR data were analyzed using the 2-ΔΔCT method accordingto the manufacturer's directions. Patient X had a proviral load of 6,100copies per 10⁶ PBMC.

Example 7 Measuring Patient T-Cell Counts

Patient X's blood was sent to LabCorp® for measurement of CD4 and CD8 Tcell levels. The CD4 T cell levels and CD4:CD8 ratios were determined byProtocols 505008 and 505271, respectively. Patient X had a CD4 T-cellcount of 338 cells/μl and a CD4:CD8 ratio of 0.52.

What is claimed is:
 1. An isolated human immunodeficiency virus type 2(HIV-2) comprising the virus deposited as HIV-2NWK08F, said virus havingATCC accession number PTA-9773.
 2. An isolated HIV-2NWK08F viruscomprising the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:4.